ABSTRACT Membrane proteins are of extreme importance for human health and 30% of the human proteome consist of membrane proteins, which are key players in important cellular processes controlling and mediating the interaction between cells, regulate transport in and out of the cells and are also the major players in bioenergy conversion including respiration; 40% of all current drugs being targeted to membrane proteins. However knowledge of their structure and function at the atomic level is sparse, as the structures for less 800 unique membrane proteins has been determined to date. Furthermore, most of these structures show only a static picture of the membrane protein, while their function in the cell is highly dynamic. This proposal aims to develop novel methods to determine ?molecular movies? of membrane proteins ?at work,? and specifically, to determine the dynamics of the catalytic cycle of the cytochrome oxidase and to study the conformational dynamics of the beta-adrenergic G-protein coupled receptor. The proposal aims to develop and use a combination of methods that focus on time-resolved femtosecond (fs) X-ray crystallography with XFELs. Fs crystallography, pioneered by our team in the previous funding cycles, has revolutionized X-ray crystallography. It overcomes radiation damage, enabling structure analysis of biological macromolecules at room temperature under physiological conditions based on the new serial approach for structure determination, where tens of thousands of X-ray diffraction snapshots are collected from a stream of fully hydrated nano/microcrystals of proteins, interacting with fs X-ray pulses from a Free Electron Laser. New developments in nanocrystal growth and characterization, together with development of new injector technology and data evaluation methods, have progressed the new method of SFX at a very fast pace based on results from this project, which has led to 70 publications, 29 of them in high impact journals as well as patents (accepted and provisional applications filed). This R01 Renewal is based on the success of the previous work but explores new areas by shifting the major focus from the proof-of-concept to their improvement and application?towards molecular movies of the functional dynamics of two important membrane proteins: cytochrome c oxidase and the beta-adrenergic receptor. In Aim 1 we will study the dynamics of the catalytic cycle of cytochrome c oxidase, a large-multi-protein membrane protein complex and the key enzyme in respiration, catalyzing the 4 electron 4 proton reduction of oxygen to 2 water molecules, whose detailed mechanisms is still a hot topic of debate. Aim 2 is focused on the dynamics of the beta-adrenergic receptor and its complexes with arrestin and G-protein. In this Aim, we will combine new SFX method developments (including making the GPCR triggered by light to allow for very fast dynamics to be detected) with the time resolved cryo-EM studies of the GPCR complexes that will allow us to unravel motions on a time scale of seconds.